Isolation and cloning of coat protein gene of rice tungro bacilliform virus (RTBV)

Abstract

Paddy is an important food crop in Malaysia. However, this plant has been attacked by diseases and various types of biotic and abiotic stresses. The two main pathogens of rice are fungi and viruses. In controlling plant viral diseases, genes such as the viral coat protein, polymerase and protease have been successfitlly used in developing transgenic crops with resistance to viruses. In producing crops with resistance to rice tungro bacilliform virus (RTBV), the coat protein has been isolated and transformed into the paddy varieties. Total genomic DNA was extracted from RTBV infected Taichung Native1 (TN-I) variety. The extracted genomic DNA was then used as a template to obtain the coat protein gene of RTB V through the polymerase chain reaction (PCR). The forward primer, 5'-ACC AAA GAA ACA CGG-3' (TM 50.JOC) and reverse primer.Y'<CI'T GAC TAG AAT GTG C-3' (SI.6DC) were used in amplifying the coat protein gene of RTBV at an annealing temperature of 51. SOe. The PCR product was electrophoresed on 1.5% agarose gel and an approximately 1000 bp band representing the coat protein gene was observed. The PCR product was then cloned into pGEM??- T Easy before being transformed into E. coli JM109 competent cell. Plasmid DNA was extracted from the putative recombinant lanes obtained. The DNA sequence obtained showed high level of similarity (96%) with the coat protein gene of RTBV